Changes In Anogenital Distance Research Articles

Gestational and pubertal exposure to low dose of di-(2-ethylhexyl) phthalate impairs sperm quality in adult mice

Di-(2-ethylhexyl)-phthalate (DEHP) is a compound widely used as a plasticizer, which can leach from plastics into the environment and thus influence human health. The aim of this study was to analyze whether exposure to an environmentally relevant dose of DEHP during mice fetal development or puberty can cause long-lasting changes detectable month/s after the last exposure. We used a DEHP concentration relevant to a daily human intake of 2.4–3 μg/kg of body weight/day. CD1 outbred mice were treated either in utero or postnatally during puberty and analyzed in adulthood. Analyzing fertility parameters using morphometric, histologic, genomic and proteomic methods we showed that DEHP exposure leads to decreased sperm concentration and quality, in both experimental groups. Moreover, the changes in anogenital distance, seminal vesicle weight, and testicular gene expression suggest a disturbance of androgen signaling in exposed animals. In conclusion, we hereby present, that the prenatal and pubertal exposure to a low dose of DEHP negatively influenced reproductive endpoints in male mice, and some of the effects were persistent until adulthood.

Anogenital Distance in Healthy Infants: Method-, Age- and Sex-related Reference Ranges.

The use of anogenital distance (AGD) in clinical and epidemiological settings is increasing; however, sex-specific reference data on AGD and data on longitudinal changes in AGD in children is scarce. To create age-, sex-, and method-related reference ranges of AGD in healthy boys and girls aged 0-24 months, to assess the age-related changes in AGD and to evaluate the 2 predominantly used methods of AGD measurement. The International AGD consortium comprising 4 centers compiled data from 1 cross-sectional and 3 longitudinal cohort studies (clinicaltrials.gov [NCT02497209]). All data were collected from population-based studies, recruiting from 4 maternity or obstetric centers (United States, Cambridge [United Kingdom], Odense, and Copenhagen [Denmark]). This study included a total of 3705 healthy, mainly Caucasian children aged 0-24 months on whom 7295 measurements were recorded. AGDAS (ano-scrotal), AGDAF (ano-fourchette), AGDAP (ano-penile), AGDAC (ano-clitoral), AGD body size indices (weight, body mass index [BMI], body surface area, and length), and intra- and interobserver biases. We created age-specific reference ranges by centers. We found that AGD increased from birth to 6 months of age and thereafter reached a plateau. Changes in AGD/BMI during the first year of life were minor (0-6% and 0-11% in boys and girls, respectively). Reference ranges for AGD can be used in future epidemiological research and may be utilized clinically to evaluate prenatal androgen action in differences-in-sex-development patients. The increase in AGD during the first year of life was age-related, while AGD/BMI was fairly stable. The TIDES and Cambridge methods were equally reproducible.

Prenatal perfluoroalkyl substances and newborn anogenital distance in a Canadian cohort

Exposure to the man-made chemicals perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS) and perfluorohexanesulfonate (PFHxS) is widespread. These perfluoroalkyl substances (PFASs) have been associated with androgenic endocrine-disrupting properties; however, the evidence is equivocal and few human studies have examined the association between prenatal exposure to PFASs and markers of androgenic endocrine disruption such as changes in anogenital distance (AGD).In the MIREC cohort, PFOA, PFOS and PFHxS were analyzed in first trimester maternal plasma. AGD was measured in 205 male and 196 female newborns. The change in estimate procedure was used to identify confounders by sex and AGD in multiple linear regression models.Geometric mean plasma concentrations (95% CI) for PFOA, PFOS and PFHxS were 1.71 (1.61, 1.81), 4.40 (4.18, 4.64) and 1.15 (1.06, 1.25) μg/L, respectively. A one-unit increase in natural log transformed PFOA was associated with a 1.36 mm (95% CI 0.30, 2.41) increase in anoscrotal distance, adjusting for household income, active smoking status during pregnancy and gestational age. However, when examined by quartiles, a non-monotonic pattern was observed with wide confidence intervals. No consistent patterns were observed between maternal PFAS concentrations and female AGDs.This study found no clear evidence that maternal plasma concentrations of PFOS, PFOA or PFHxS were associated with shorter infant anogenital distance in males or any change in AGD in females. Whether the positive association observed between longer anoscrotal distance and PFOA is real or would have any long-lasting effect on the reproductive health of males is unknown and needs to be investigated further.

High Prenatal Exposure to Bisphenol A Reduces Anogenital Distance in Healthy Male Newborns.

Objective:To estimate the relationship between cord blood bisphenol A (BPA) levels and anogenital measurements in healthy newborns.Methods:Pregnancy and birth history, together with body mass and length data, anogenital measurements, penile measurements and cord blood samples were obtained from healthy newborns. Cord blood concentration of BPA was analyzed by sandwich enzyme-linked immunosorbent assays kit.Results:Among 130 healthy newborns (72 boys, 58 girls), mean anopenile distance was 45.2±6 mm and anoscrotal distance was 21.9±5.4 mm in boys; mean anoclitoral distance was 33.8±6.6 mm and mean anofourchette distance was 12.2±4.9 mm in girls. Mean cord blood BPA level was 4.75±2.18 ng/mL. 90th percentile value for cord blood BPA was 8.26 ng/mL and the analysis showed a statistically significant correlation between anoscrotal distance and cord blood BPA levels above the 90th percentile (p=0.047) in boys. The changes in anogenital distance in girls were not statistically significant.Conclusion:We showed a significant association between high cord blood BPA levels and shortened anoscrotal distance in male newborns. However, this result should be interpreted with caution since there were no significant external genital abnormalities in our study group.

Synergistic Disruption of External Male Sex Organ Development by a Mixture of Four Antiandrogens

BackgroundBy disrupting the action of androgens during gestation, certain chemicals present in food, consumer products, and the environment can induce irreversible demasculinization and malformations of sex organs among male offspring. However, the consequences of simultaneous exposure to such chemicals are not well described, especially when they exert their actions by differing molecular mechanisms.ObjectivesTo fill this gap, we investigated the effects of mixtures of a widely used plasticizer, di(2-ethylhexyl) phthalate (DEHP); two fungicides present in food, vinclozolin and prochloraz; and a pharmaceutical, finasteride, on landmarks of male sexual development in the rat, including changes in anogenital distance (AGD), retained nipples, sex organ weights, and malformations of genitalia. These chemicals were chosen because they disrupt androgen action with differing mechanisms of action.ResultsStrikingly, the effect of combined exposure to the selected chemicals on malformations of external sex organs was synergistic, and the observed responses were greater than would be predicted from the toxicities of the individual chemicals. In relation to other hallmarks of disrupted male sexual development, including changes in AGD, retained nipples, and sex organ weights, the combined effects were dose additive. When the four chemicals were combined at doses equal to no observed adverse effect levels estimated for nipple retention, significant reductions in AGD were observed in male offspring.ConclusionsBecause unhindered androgen action is essential for human male development in fetal life, these findings are highly relevant to human risk assessment. Evaluations that ignore the possibility of combination effects may lead to considerable underestimations of risks associated with exposures to chemicals that disrupt male sexual differentiation.

Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat.

ObjectiveThe aim of this study was to assess whether the joint effects of three androgen receptor antagonists (vinclozolin, flutamide, procymidone) on male sexual differentiation after in utero and postnatal exposures can be predicted based on dose–response data of the individual chemicals.MethodsTest chemicals and mixtures were administered by gavage to time-mated nulliparous, young adult Wistar rats from gestational day 7 to the day before expected birth, and from postnatal days 1–16. Changes in anogenital distance (AGD) and nipple retention (NR) in male offspring rats were chosen as end points for extensive dose–response studies. Vinclozolin, flutamide, and procymidone were combined at a mixture ratio proportional to their individual potencies for causing retention of six nipples in male offspring.ResultsWith AGD as the end point, the joint effects of the three anti-androgens were essentially dose additive. The observed responses for NR were slightly higher than those expected on the basis of dose addition. A combination of doses of each chemical, which on its own did not produce statistically significant AGD alterations, induced half-maximal mixture effects. At individual doses associated with only modest effects on NR, the mixture induced NR approaching female values in the males.ConclusionsEffects of a mixture of similarly acting anti-androgens can be predicted fairly accurately on the basis of the potency of the individual mixture components by using the dose addition concept. Exposure to anti-androgens, which individually appears to exert only small effects, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.

Diisobutyl phthalate has comparable anti-androgenic effects to di- n-butyl phthalate in fetal rat testis

Phthalates are widely used as plasticizers in various consumer products and building materials. Some of the phthalates are known to interfere with male reproductive development in rats, and di- n-butyl phthalate (DBP), diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) were recently banned for use in toys in the EU mainly due to their reproductive toxicity. Diisobutyl phthalate (DiBP) has similar structural and application properties as DBP, and is being used as a substitute for DBP. However, knowledge on male reproductive effects of DiBP in experimental animals is lacking. Methods In the current study, four groups of pregnant Wistar rats were exposed to either 0 mg/kg bw/day or 600 mg/kg bw/day of DiBP from gestation day (GD) 7 to either GD 19 or GD 20/21. Male offspring was examined at GD 19 or GD 20/21 for effects on testicular testosterone production and testicular histopathology. Changes in anogenital distance (AGD) were evaluated as an indication of feminisation of males. Results Anogenital distance was statistically significantly reduced at GD 20/21 together with reductions in testicular testosterone production and testicular testosterone content. Histopathological effects (Leydig cell hyperplasia, Sertoli cell vacuolisation, central location of gonocytes and presence of multinuclear gonocytes) known for DBP and DEHP were observed in testes of DiBP-exposed animals at GD 20/21. Additionally, immunohistochemical expression of P450scc and StAR proteins in Leydig cells was reduced by DiBP. At GD 19, these effects on anogenital distance, testosterone levels and histopathology were less prominent. Conclusion In this study, GD 20/21 rather than GD 19 appears to be the optimal time for investigating changes in anogenital distance, testosterone levels, and testicular histopathology. DiBP has similar testicular and developmental effects as DBP and DEHP, and although more developmental and especially postnatal studies are needed to clearly identify the reproductive effects of DiBP, this study indicates a reason for concern about the use of DiBP as a substitute for DBP.

Male reproductive tract lesions at 6, 12, and 18 months of age following in utero exposure to di(n-butyl) phthalate.

In utero exposure of male rats to the antiandrogen di(n-butyl) phthalate (DBP) leads to decreased anogenital distance (AGD) on postnatal day (PND) 1, increased areolae retention on PND 13, malformations in the male reproductive tract, and histologic testicular lesions including marked seminiferous epithelial degeneration and a low incidence of Leydig cell (LC) adenomas on PND 90. One objective of this study was to determine the incidence and persistence of decreased AGD, increased areolae retention, and LC adenomas in adult rats following in utero DBP exposure. A second objective was to determine whether AGD and areolae retention during the early postnatal period are associated with lesions in the male reproductive tract. Pregnant Crl:CD(SD)BR rats were gavaged with corn oil or DBP at 100 or 500 mg/kg/day, 10 dams per group. Three replicates of rats (n = 30 rats per replicate) were exposed from gestation day 12 to 21 and the male offspring allowed to mature to 6, 12, or 18 months of age. Gross malformations in the male reproductive tract and histologic lesions in the testes were similar to those previously described. However, testicular dysgenesis, a lesion of proliferating LCs and aberrant tubules that has not been previously described in DBP-exposed testes, was diagnosed. The incidence of this lesion was approximately 20% unilateral and 7-18% bilateral in the high-dose group and was similar among all ages examined, implicating a developmental alteration rather than an age-related change. AGD and areolae retention were found to be permanent changes following in utero exposure to 500 mg/kg/day of DBP. Decreased AGD was a sensitive predictor of lesions in the male reproductive tract, relatively small changes in AGD were associated with a significant incidence of male reproductive malformations. In utero DBP exposure induced proliferative developmental lesions, some of which would have been diagnosed as LC adenomas by the morphological criteria set forth by the Society of Toxicologic Pathology. However, these lesions were dissimilar to traditional LC adenomas as the LCs were poorly differentiated and the lesions contained aberrant seminiferous tubules. While the morphology and incidence of this DBP-induced testicular developmental lesion has been fully characterized by this study, the detailed pathogenesis warrants further investigation.

Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat.

Finasteride is a specific inhibitor of type II 5alpha-reductase, the enzyme that converts testosterone (T) to the more potent androgen receptor agonist dihydrotestosterone (DHT). In utero exposure to androgen receptor antagonists and T biosynthesis inhibitors have induced permanent effects on androgen-sensitive end points such as anogenital distance (AGD), nipple retention, and malformations of the male rat reproductive tract. The objectives of this study were to (1) characterize the dose response of finasteride-mediated alterations in androgen-dependent developmental end points, (2) determine whether prenatal exposure to finasteride permanently decreases AGD or results in nipple retention, and (3) evaluate whether AGD or nipple retention is predictive of adverse alterations in the male reproductive tract. Pregnant Crl:CD(SD)BR rats (n=5-6/group) were gavaged with either vehicle or finasteride at 0.01, 0.1, 1.0, 10, or 100 mg/kg/day on gestation days 12 to 21. All male offspring were monitored individually until necropsy on postnatal day (PND) 90. The present study design has been used previously for other antiandrogens and is sensitive to perturbations of the male rat reproductive tract. Decreases in AGD on PND 1 and increases in areolae-nipple retention on PND 13 were significantly different from controls in all finasteride-exposed male rats. Finasteride-induced changes in AGD and nipple retention were permanent in male rats exposed to finasteride at and above 0.1 mg/kg/day. On PND 90, dorsolateral and ventral prostate lobes were absent in 21 to 24% of rats exposed to 100 mg/kg/day finasteride and weighed significantly less at and above 10 mg/kg/day. In the highest dose group, 73% of animals had ectopic testes, much higher than previously reported. The most sensitive malformation other than decreased AGD and nipple retention was the dose-dependent increase in hypospadias. The lowest observed adverse effect level (LOAEL) for finasteride-induced permanent effects in this study was 0.1 mg/kg/day based on permanent changes in AGD and nipple retention. Finasteride-induced changes in AGD and retention of nipples were highly predictive of hypospadias, ectopic testes, and prostate malformations even though some animals with retained nipples or decreased AGD may not have had other reproductive tract malformations. In summary, prenatal exposure to finasteride specifically inhibited DHT-mediated development with little to no change in T-mediated development.

Male rats exposed to linuron in utero exhibit permanent changes in anogenital distance, nipple retention, and epididymal malformations that result in subsequent testicular atrophy.

Prenatal exposure to the herbicide linuron, a weak androgen receptor antagonist, has been shown to perturb androgen-dependent male rat reproductive development as evidenced by slight decreases in anogenital distance (AGD), increased retention of areolae/nipples, and induction of epididymal malformations in combination with testicular atrophy in the adult rat over dose levels ranging from 12.5 to 100 mg/kg/day. Studies were undertaken to determine whether linuron-mediated changes in AGD and nipple retention are permanent, whether linuron is a direct testicular toxicant, and if there was an association between areola/nipple retention and malformations. Pregnant rats were administered corn oil vehicle or linuron by gavage at 0 or 50 mg/kg/day (n = 8 controls, 20 treated) from gestation days 12 to 21. Male offspring were necropsied on postnatal days (PND) 35 and 56. Linuron-exposed male rats exhibited a significant (8%) decrease in AGD on PND 1 and a similar decrease was also observed on PND 56. Linuron-exposed male rats displayed an increase in areola retention on PND 13, as evidenced by 0.6 +/- 0.5 and 3.3 +/- 0.4 areolae per rat in the control and exposed groups, respectively. Male rats displayed a significant increase in nipple retention on PND 35 and 56 (collectively) of 0 +/- 0.5 and 1.7 +/- 0.3 nipples per rat in control and exposed groups, respectively. On PND 35, 4/51 rats (3/9 litters) from linuron-treated dams displayed enlarged testes in combination with malformed epididymides. Epididymal malformations were observed in 19/51 rats (6/9 litters) in the linuron-exposed dose group. On PND 56, grossly enlarged and edematous testes were seen in 16/56 linuron-exposed rats (6/9 litters). Epididymal lesions were observed in 23/58 rats (6/9 litters). Microscopically, all linuron-exposed animals that exhibited a testicular lesion on PND 56 also displayed an epididymal lesion. These lesions were not seen in control animals. Approximately 25 and 60% of the male offspring that had malformations of the epididymis and vas deferens did not exhibit either areolae on PND 13 or nipples at necropsy, respectively. These data indicate that in utero linuron exposure to 50 mg/kg/day results in permanent changes in AGD and nipple retention in male rats. Moreover, these findings indicate that linuron-induced testicular atrophy, which is observed in adult rats, is secondary to increased intratubular pressure resulting from obstruction of testicular fluid outflow subsequent to malformation of the epididymides. These data also suggest that although linuron-mediated retention of areolae on PND 13 and nipples at necropsy may be suggestive of altered testosterone-mediated reproductive development seen in adult rats, these endpoints are not predictive.

Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues.

Male offspring exposed in utero to antiandrogens often display alterations in androgen-dependent developmental markers (e.g., anogenital distance [AGD], nipple retention) together with clearly adverse responses such as genital malformations and reproductive tract lesions. The objectives of this study were to determine whether in utero exposure to flutamide results in permanent changes in male AGD and nipple retention, characterize the dose-response relationship between flutamide-mediated alterations in these landmarks and clearly adverse antiandrogenic effects, and establish the predictive value and relationship between AGD and nipple retention, and other adverse manifestations. Male offspring were exposed in utero to 0, 6.25, 12.5, 25, or 50 mg/kg/day (po) of flutamide from gestation days 12 to 21. Offspring were uniquely identified at birth, and various androgen-mediated end points (AGD, areola/nipple retention, cryptorchidism, reproductive tract weights, and malformation incidence) were examined throughout life. In utero flutamide exposure significantly decreased the AGD on postnatal day (PND) 1 and increased areola/nipple retention in male rats on PND 13. Flutamide-induced alterations in AGD and areolae/nipples in early postnatal life correlated with a reduction in AGD and retained nipples observed in the adult. Prenatal flutamide exposure resulted in dose-responsive increases in cryptorchidism. Hypospadias were observed in all flutamide-exposed offspring. In utero flutamide exposure induced partial or complete prostate agenesis and decreased the weights of the seminal vesicles, levator ani bulbocavernosus (LABC) muscle, testes, and epididymides in a dose-dependent manner. Epididymal malformations were observed mainly in the 50 mg/kg/day flutamide dose group. In general, flutamide-induced alterations in dihydrotestosterone (DHT)- and testosterone (T)-dependent development each had similar respective dose-response curves. DHT-mediated development was more sensitive to in utero flutamide exposure than T-dependent processes. However, the dose-response curves for flutamide-induced changes in cryptorchidism and seminal vesicle weight were intermediate between the dose-response curves for DHT- and T-mediated development, indicating that proper development of these tissues may require both androgens. The LABC also displayed a dose-dependent decrease in weight that was similar to dose-response observed with seminal vesicle weight and was the most sensitive T-dependent end point measured. Flutamide-induced decreases in AGD predicted subsequent malformations as evidenced by logistic regression and receiver operator characteristic analysis of malformations versus AGD. However, the AGD that would predict a 10% incidence of seminal vesicle malformations is equivalent to a female AGD. An almost fully feminized phenotype of 10-12 nipples was observed in animals that had malformations in T-dependent tissues, whereas 6 or more nipples were observed in animals with malformation in DHT-dependent tissues. These data suggest that flutamide-mediated changes in AGD and nipple retention are not sensitive predictors of altered T-mediated development.

TWO-GENERATION REPRODUCTIVE TOXICITY STUDY OF TRIBUTYLTIN CHLORIDE IN FEMALE RATS

A two-generation reproductive toxicity study of the effects of tributyltin chloride (TBTCl) was conducted in female rats using dietary concentrations of 5, 25, and 125 ppm TBTCl. Reproductive outcomes of dams (number and body weight of pups and the percentage of live pups) and the growth of female pups (the day of eye opening and body weight gain) were significantly decreased in the 125 ppm TBTCl group. A delay in vaginal opening and impaired estrous cyclicity were also observed in the 125 ppm TBTCl group. However, an increase in anogenital distance was found in all TBTCl groups on postnatal d 1. A dose-effect relationship was observed in TBTCl-induced changes in anogenital distance. These results indicate that the whole-life exposure to TBTCl affects the sexual development and reproductive function of female rats. In addition, the TBTCl-induced increase in anogenital distance seems to suggest it may exert a masculinizing effect on female neonates. However, the concentrations of TBTCl used in this study are not environmentally relevant.